The c-Met receptor kinase regulates cellular proliferation, migration, differentiation and branching morphogenesis during development and homeostasis1-3. Met is also expressed on the cell surface of a variety of human primary solid tumors and in their metastases (www.vai.org/met/). The amino acid sequence of the extracellular domain of human Met is provided in SEQ ID NO:1, amino acids 25-567. In its activated state, the Met receptor controls growth, invasion, and metastasis of cancer cells through multiple signal transduction pathways4. In some cancer cell lines, loss of Met expression through silencing promotes apoptosis, demonstrating that Met is necessary for survival5-7. Met activity increases through mutations in the kinase or juxtamembrane domains8-11, through overexpression12, 13, or through binding to its ligand, hepatocyte growth factor (HGF/SF)14, 15. Activating mutations of Met in the germline, which stimulate ligand-independent Met activation, are the cause for development of hereditary papillary renal carcinomas10. In cancerous cells, selective amplification of the mutated Met allele further enhances overall Met kinase activity16. The magnitude of Met expression predicts the aggressiveness of a number of cancer types (www.vai.org/met/). Accurate detection and quantification of Met protein expression are needed to identify cancers that are likely responsive to Met inhibitors and the development of such molecular diagnostics lags significantly behind the drug development.
High level expression of c-Met has been associated with poor prognosis in many cancer types, including breast, gastric, cervix, hepatocellular and head and neck17-22. Approximately 25% of ovarian cancers and 11% of gliomas express high levels of c-Met. In breast cancer, c-Met expression is observed in a subset of cancers independent of Her2, but is associated with increased cell proliferation23,24. In ductal breast cancer, simultaneous expression of Syndecan-1, E-cadherin and c-Met enhances angiogenesis and lymphangiogenesis25. Any disease associated with c-Met expression is referred to herein as a “Met-related disease”. However, the reliability of the immunohistochemical studies has been questioned in light of the lot-to-lot variability of the antiserum raised against the C-terminal peptide in the c-Met receptor, that has been used in most studies26. Most commercially available Met antibodies are unreliable (or have not been stringently tested for their reliability). In addition to increased c-Met expression, elevated HGF/SF concentrations in the tumor microenvironment have also been associated with adverse outcome. For example hypoxic tumor stromal cells in pancreatic cancer increase HGF secretion and accelerate pancreatic cancer progression27.
Mesenchymal cell lines with engineered c-Met and HGF expression are highly metastatic and expression of mutant c-Met receptor or amplification of the c-Met locus in cells lines increases the proliferative, invasive and metastatic phenotype of cancers6, 28-30. Together with Myc, wild type c-Met causes mammary carcinogenesis31. c-Met is one of the most frequently genetically altered or otherwise dysregulated receptor tyrosine kinases (RTK) in advanced human cancers and thus represents an attractive treatment target. Kinase activating c-Met mutations are observed in sporadic renal, lung, head and neck, hepatocellular carcinoma, non small cell lung cancer (NSCLC), gastric cancer and melanoma13, 32-35. Furthermore, amplification of the c-Met locus has been detected in gastric, metastatic colorectal and esophageal adenocarcinoma12, 13, additional Met-related diseases. Activation of c-Met in cancer cells induces the secretion of angiogenic factors, such as VEGFA and IL-8 and inhibits synthesis of thrombospondin-1, an anti-angiogenic factor36, 37. In addition, c-Met activation in endothelial cells causes angiogenesis. While the cytotoxic effects of inhibiting Met activity may only occur in cancers with activated c-Met, the antiangiogenic effect may exist more frequently.
A major advance occurred with the development of small molecule inhibitors of c-Met that are orally bioactive (“Met-inhibiting agents”). Of these inhibitors, PF-2341066 demonstrates specificity for inhibition of c-Met and anaplastic lymphoma kinase (ALK) and leads to regression of of GTL-16 gastric cancer xenografts and NCI-H441 NSCLC xenografts at a dose of 50 mg/kg/day38. At this dose, c-Met is completely inhibited and maximal drug efficacy with long duration is achieved. Preclinical studies with one-armed anti-c-Met antibodies and small molecule kinase inhibitors7, 39, 40 as well as early clinical studies in patients further highlight the promise of c-Met inhibitors against a variety of cancer types, their favorable pharmacodynamic properties and low toxicity. Thus, when used to treat cancers with an active Met axis, these drugs may indeed benefit many cancer patients. However, the molecular diagnostic tools to identify cancers that possess active Met pathways are not available.
Molecular diagnostics to detect expression and determine the activation state of treatment targets of kinase inhibitors are urgently needed to improve the treatment of cancer patients. Drugs that bind cell surface receptors or permeate into cells and inhibit receptor and non-receptor kinases show immense promise in the clinic, however the detection of the corresponding targets in human cancers provides a major challenge. Besides the Hercept test for quantitative measurement of Her-2/Neu expression, which required a lengthy and arduous development for use in routine clinical samples and FDA approvement, no validated diagnostic tests for receptor or non-receptor protein kinase expression are available. The difficulty in developing these diagnostic reagents stems from the low level expression of kinases, the labile activation state, which depends on protein phosphorylation and the poor specificity of antibodies against most phospho-epitopes, which indicate kinase activity. Consequently, most receptor tyrosine kinases (RTK) lack detection reagents for expression measurements in formalin-fixed paraffin embedded (FFPE) tissues, which is the most commonly obtained tissue preparation from patient cancers. It is becoming increasingly obvious that patient stratification for treatment with kinase inhibitors is a crucial for success with the anti-neoplastic activity of this group of agents. For a given solid tumor type, the frequency of cancers expressing the drug responsive target protein is small. Thus, if patients are not carefully selected for treatment, many agents could fail to demonstrate efficacy in phase II and phase III clinical trials.
The c-Met receptor is particularly difficult to measure in FFPE tissues because of poor reagent choices, insufficient validation of the performance of c-Met antibodies in FFPE tissues and sensitivity of c-Met to formalin-fixation. Given the promise of novel c-Met inhibitory agents in the clinic, companion diagnostics are needed to identify patients who would potentially benefit from these agents.